We found that outpatient SARS-CoV-2 variant surveillance could be conducted in a private laboratory in a timely and accurate manner. SARS-CoV-2 positivity rates from specimens tested in a private laboratory were similar to county level data collected during the same periods. Among a sample of people that tested positive for SARS-CoV-2, we observed that initially the Alpha variant of SARS-CoV-2 was most prevalent in Los Angeles and Riverside County in May 2021, however the Delta variant of SARS-CoV-2, mainly the AY.4 and AY.12 strains, became dominant over a period of weeks. Among the isolates identified as a SARS-CoV-2 Delta variant, a large number of isolates carried mutations classifying them as a sub-lineages of Delta, which is evidence of continued mutation. When compared to publicly available data on SARS-CoV-2 variants, our surveillance sampling found similar proportions of variants of concern in our outpatient samples over time.23
After chronic neglect of defunding, the public health infrastructure of the United States was ill equipped for the COVID-19 pandemic.24 Despite earlier warnings of SARS-CoV-2 epidemics in early 2020,25,26 the United States was not prepared for SARS-CoV-2 testing.4 Despite the poor state of public health infrastructure of the United States for a pandemic, hundreds of thousands of deaths could have potentially been avoided if public-private partnerships were developed for make up for the dearth in diagnostic testing and health workforce capacity.27
The identification of new SARS-CoV-2 variants in a timely manner is critical to public health. While it is hard to prognosticate the future, it is possible to establish a method to prioritize research when new mutations are discovered on genetic coding segments of key proteins, like the SARS-CoV-2 spike protein.28,29 Faster identification of new SARS-CoV-2 variants of concern and understanding the rates in their change of prevalence could be critical predictors of new waves of SARS-CoV-2 and met with changes in public health recommendations. This study demonstrates that private laboratories have a role in the surveillance of SARS-CoV-2 variants of concern.
Temporally, it can be observed that the Delta variant of SARS-CoV-2 arose following en masse vaccination efforts. There are many reported cases of breakthrough SARS-CoV-2 infections among people who are fully vaccinated. Many of those people had high viral loads (cycle threshold values less than 30), and with cycle threshold values similar when comparing those who were vaccinated to those who were not vaccinated.30,31 Reassuringly, most breakthrough infections were mild or asymptomatic.32 Additionally, only small differences were observed in vaccine effectiveness against symptomatic disease or death when comparing the Delta to Alpha variant with the BNT162b2 vaccine (93.7% with Alpha and 88.0% for Delta) and ChAdOx1 nCoV-19 vaccine (74.5% with Alpha and 67.0% for Delta).33 Due to the erosion gf vaccine efficacy against the SARS-CoV-2 Delta variant, it is possible that vaccinations may have played a role in the selective pressure for the Delta variant prevalence in areas with high vaccination rates.12,14,29,34
In our efforts to monitor SARS-CoV-2 variants of concern, we sequenced a non-identifiable isolate of SARS-CoV-2, hCoV-19/USA/CA-Curative-707962712299/2021. The non-identifiable isolate simultaneously carried classic S protein mutations present in the Delta variant, while also displaying hallmark S protein mutations observed in Alpha, Beta, Mu and Kappa variants of SARS-CoV-2. The non-identifiable isolate carried seven S protein mutations prevalent in Mu variant, notably the S:R346K amino acid substitution in the Receptor Binding Domain and two mutations of concern/interest, S:E484K and S:N501Y, in the Receptor-Binding Motif,35 which are also normally absent in Delta and Delta plus variants. Also, the non-identifiable isolate carried 7 mutations prevalent in AY.20 sub-lineage of Delta, including P681R mutation special for Delta lineage and known to facilitate the spike protein cleavage, enhance cell-level infectivity and pathogenicity.28 It carried other common Delta mutations (S:T19R, S:T478K) that are not prevalent in Mu variants. Notably, the isolate carries mutations affecting epitopes for all three main classes of neutralizing antibodies (Class 1-N501Y, T478K; Class 2-E484K, L452; and Class 3-R346K), which brings concerns that this isolate might have evolutionary advantages similar to the Delta variant of SARS-CoV-2 with its ability to evade the immune system of vaccinated persons and have increased infectivity.12,36
The sheer number of people who have been infected and the total SARS-CoV-2 infected person-time has led to the rapid evolution of SARS-CoV-2. Local epidemics of populous areas creates a situation in which many new mutations can form due to the large amount of viral replication over a short period of time. It is essential that all global SARS-CoV-2 epidemics are controlled to limit the rate of new SARS-CoV-2 mutations.
The study had the following limitations. Given that our study population consisted of people undergoing outpatient SARS-CoV-2 testing, it is possible that the variants we identified are less likely to cause critical illness if those patients required hospitalization. The study was conducted in Los Angeles and Riverside Counties therefore temporal changes may differ from other outbreaks across the country and world. Reclassification of SARS-CoV-2 variants may change our results: When identifying the Delta strain of SARS-CoV-2, some of the Delta lineages might be further mutated sub-lineages of Delta that carry hallmark mutations of the Delta variant not covered well in generated consensuses. When re-running our samples though the latest version of PangoLearn, sub-lineages of the Delta variant were observed in our study sample since 2020. These samples were not identified as special sub-lineages or variants of concern until variants were reclassified. It is likely that the isolates we sequenced will be further reclassified as new lineages or have features of interest/concern as governing bodies determine new SARS-CoV-2 variants of concern in the future.